Method of genetic modification of a wild type viral sequence

ABSTRACT

The present invention concerns a method of genetic modification of a TGB-3 wild type viral sequence for reducing or suppressing the possible deleterious effects of the agronomic properties of a transformed plant or plant cell by said TGB-3 viral sequence, comprising the following successive steps: submitting said sequence to point mutation(s) which allow the substitution of at least one amino-acid into a different amino-acid; selecting genetically modified TGB-3 wild type viral sequences having said point mutation(s) and which are not able to promote cell-to-cell movement of a mutant virus having a dysfunctional TGB-3 wild type viral sequence, when expressed in trans from a replicon; further selecting among said genetically modified TGB-3 viral sequences, the specifically genetically modified sequence which inhibits infection with a co-inoculated wild type virus when the mutant form was expressed from a replicon; and recovering said specifically genetically modified TGB-3 viral sequence.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/743,905, filed Apr. 24, 2001, the entire disclosure of which ishereby expressly incorporated by reference. The U.S. patent applicationSer. No. 09/743,905, filed Apr. 24, 2001, is the U.S. National Phaseunder 35 U.S.C. §371 of International Patent Application PCT/BE99/00089,filed Jul. 9, 1999 designating the U.S. and published in English on Jan.20, 2000 as WO 00/03025, which claims priority of European patentapplication EP 98870159.5, filed Jul. 10, 1998.

FIELD OF THE INVENTION

The present invention is related to a method of genetic modification ofa wild type viral sequence, for reducing or suppressing deleteriousproperties of plants or plant cells transformed by said wild type viralsequence.

The present invention is also related to the modified viral sequenceobtained by said method, and to the plant and the plant cell comprisingsaid modified viral sequence.

BACKGROUND OF THE INVENTION

The widespread viral disease of the sugar beet plant (Beta vulgaris)called Rhizomania is caused by a furovirus, the beet necrotic yellowvein virus (BNYVV) (Tamada T. & Baba T. 1973 Annals of thePhytopathological Society of Japan 39:325-332; Kuszala M. & Putz C. 1977Annals of Phytopathology 9:435-446) which is transmitted to the root ofthe beet by the soil borne fungus Polymyxa betae (Keskin B. 1964 Archivfür Mikrobiology 49:348-374).

The disease affects significantly acreages of the area where the sugarbeet plant is grown for industrial use in Europe, USA and Japan and isstill in extension in several places in Western Europe (Asher M. J. C.1993 “Rhizomania” In: The sugar beet crop, ed. D. A. Cooke and R. K.Scott, Chapman & Hall, London, pp. 312-338; Richard-Molard M. 1995Rhizomanie In Institut français de la betterave industrielle.Compte-rendu des travaux effectues en 1994, ITB, Paris pp. 225-229).

Since 1986, a number of reports and publications have described the useof isolated viral nucleotide sequences expressed in plants to confer ahigh level of tolerance against a specific infectious virus or even toconfer a broad spectrum type of resistance against a number of relatedviruses (Powell A. P. et al. 1986 Science 232:738-743; Fritchen J. H. &Beachy R. N. 1993 Ann Rev Microbiol 47:739-763; Wilson T. M. A. 1993PNAS USA 90:3134-3141). One of the most documented viral resistancestrategies based on genetic engineering, in many cultivated species suchas potato, squash, cucumber or tomato, is the use of the viralnucleotide sequence which under the control of plant regulatoryelements, encodes the coat-protein of the target virus (Gonsalves D. &Slightom, J. L. 1993 Seminars in Virology 4:397-405).

However, in coat-protein mediated resistance, the expression of acertain level of resistance in the transgenic plant might be attributedto different mechanisms such as RNA co-suppression and not necessarilyto the production of the protein sequence.

In general, the virus sequence will be transformed in an appropriatecell or tissue culture of the plant species using an Aerobacteriummediated transformation system or a direct gene transfer methodaccording to the constraints of the tissue culture or cell culturemethod which can be successfully applied in a given species. A wholeplant will be regenerated and the expression of the transgene will becharacterized.

Though sugar beet is known as a recalcitrant species in cell culture,limiting the extent of practical genetic engineering applications inthat species, there are number of isolated reports of successfultransformation and regeneration of whole plants (38). A few examples ofengineering tolerance to the BNYVV by transforming and expressing theBNYVV coat-protein sequence in the sugar beet genome have also beenpublished (Kallerhof, J. et al. 1990 Plant Cell Reports 9:24-228; WO91/13159) though they rarely report data-on whole functional transgenicsugar beet plants (Ehlers U. et al. 1991 Theoretical and Applied Genetic81:777-782). In particular, reports show limited data on the level ofresistance observed in infected conditions with transgenic sugar beetplants transformed with a gene encoding a BNYVV coat-protein sequence(Kraus J. et al. 1994 Field performance of transgenic sugar beet plantsexpressing BNYVV coat protein plants, Fourth International Congress ofPlant Molecular Biology, Int. Soc. for Plant Molecular Biology,Amsterdam; Maiss E. et al. 1994 Proceedings of the Third InternationalSymposium on the Biosafety Results of Field Tests of GeneticallyModified Plants and Microorganisms, Monterey, pp. 129-139).

A complete technology package including a transformation method and theuse of the sugar beet expression of the BNYVV coat-protein sequence asresistance source in the transgenic sugar beet plant obtained by saidtransformation method has been described in the Patent Application WO91/13159.

Based on the information published, it can not be concluded that thecoat-protein mediated resistance mechanism provides any potential forconferring to the sugar beet plant a total immunity to theBNYVV-infection by inhibiting multiplication and the virus diffusionmechanisms completely. To identify a resistance mechanism whichsignificantly blocks the spread of the virus at the early stage of theinfection process would be a major criterion of success to develop sucha transgenic resistance. In addition, such resistance would diversitythe mechanisms of resistance available.

Because the disease is shown to expand in many countries or areas, at aspeed depending upon the combination of numerous local environmental andagricultural factors, there is a major interest in diversification andimprovements of the genetic resistance mechanisms which may, alone or incombination, confer a stable and long lasting resistance strategy in thecurrent and future varieties of sugar beet plants which are grown forindustrial use.

The genome of beet necrotic yellow vein furovirus (BNYVV) consists offive plus-sense RNAs, two of which (RNAs 1 and 2) encode functionsessential for infection of all plants while the other three (RNAs 3, 4and 5) are implicated in vector-mediated infection of sugar beet (Betavulgaris) roots. Cell-to-cell movement of BNYVV is governed by a set ofthree successive, slightly overlapping viral genes on RNA 2 known as thetriple gene block (TGB), which encode, in order, the viral proteins P42,P13 and P15 (gene products are designated by their calculated Mr inkilodalton).

In the following description, the TGB genes and the correspondingproteins will be identified by the following terms: TGB-1, TGB-2, TGB-3or by their encoded viral protein number P42, P13 and P15. TGBcounterparts are present in other furoviruses and in potex-, carla- andhordeiviruses (Gilmer et al. 1992 Virology 189:40-47; Richards & Tamada1992 Annu Revendication Phytopathol 30:291-313; Bouzoubaa et al. 1987 JGen Virol 68:615-626; Herzog et al. 1994 J Gen Virol 18:3147-3155; Scottet al. 1994 J Gen Virol 75:3561-3568; Koonin & Dolja 1993 CritRevendication Biochem and Mol Biol 28:375-430). In the enclosed Table 1are represented viruses having a TGB-3 sequence, the molecular weight ofTGB-3 of said viruses, their host and references.

It has been shown previously that independent expression of P15 from aviral-RNA replication species known as a “replicon,” derived from BNYVVRNA 3, inhibits infection with BNYVV by interfering cell-to-cellmovement (Bleykasten-Grosshans et al. 1997 Mol Plant-Microbe Interact10:240-246).

In order to introduce a virus comprising a TGB-3 nucleic acid sequenceinto a plant cell or a plant, it has been proposed to incorporate anucleic acid construct comprising said TGB-3 nucleic acid sequenceoperably linked to one or more regulatory sequences active in said plant(WO 98/07875).

However, while expression of wild type TGB-3 viral sequence in atransgenic plant allows the blocking of said viral infection, thepresence of said wilt type sequence may induce deleterious effects onthe agronomic properties of transformed plants or plant cells.

Aims of the Invention

The present invention aims to provide a new method for inducing agenetic modification of a wild type viral sequence involved in themultiplication and diffusion mechanisms of virus infecting plants, inorder to reduce or suppress the possible deleterious effects upon plantsor plant cells transformed by said viral sequence.

Another aim of the present invention is to provide a method to obtainsuch a modified viral sequence which blocks virus infection when it isincorporated into a plant or a plant cell.

SUMMARY OF THE INVENTION

The present invention is related to a method of genetic modification ofTGB-3 wild type viral sequence, preferably the BNYVV P15 viral sequence,for reducing or suppressing the possible deleterious effects on theagronomic properties of the transformed plants or plant cells by saidTGB-3 viral sequence.

Preferably, said genetic modification is a point mutation which allowsthe substitution of at least one amino-acid into another differentamino-acid of said TGB-3 wild type sequence, preferably the substitutionof at least one amino-acid into another different amino-acid in theBNYVV P15 sequence.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

It seems that the function of the TGB-3 wild type sequence incell-to-cell movement involves at least in part “bridging” interactionsbetween an element of the host plant (preferably a component of theplasmodesmata), and an element of viral origin (preferably another viralprotein involved in cell-to-cell movement). Disruption of either thedomain of the TGB-3 wild type sequence (which putatively interacts withthe host element) or the domain of the TGB-3 wild type sequence (whichputatively interacts with the viral element), allows the inhibition ofthe cell-to-cell movement.

In addition, it seems that said specific mutations in wild TGB-3 allowthe type a sequence production of mutants produced in a transgenicplant, which will still interact with the viral element, but not withthe host element. These mutants might compete for binding sites on theviral element of the TGB-3 wild type sequence produced in the initialstage of the viral infection, and abort the infection by inhibitingviral movement to an adjacent cell.

Advantageously, the substitution of at least one amino-acid into anotherdifferent amino-acid of said sequence is made in regions rich inhydrophilic amino-acids usually present at the surface of the protein inits native configuration.

Preferably, the point mutation(s) allow the substitution of one or twoamino-acids into one or two different amino-acids.

In the enclosed Table 1, preferred examples of said viruses having aTGB-3 wild type viral sequence, the molecular weight of thecorresponding TGB-3 peptide, their hosts and a reference, are described.The specific wild type P15 nucleotide and amino-acid sequences of BNYVVare also already described (Bouzoubaa et al. 1986 J Gen Virol67:1689-1700). TABLE 1 Size of Virus TGB-3 Host Reference Apple stem  8kDa apple Jelkman, 1994 J Gen Virol pitting virus 75:1535-1542 Blueberry 7 kDa blueberry Cavileer et al. 1994 J Gen Viol scorch virus 75:711-720Potato virus  7 kDa potato Zavriev et al. 1991 J Gen Virol M 72:9-14White clover  8 kDa clover Forster et al. 1988 Nucl Acids Res mosaicvirus 16:291-303 Cymbidium 10 kDa orchid Neo et al. 1992 Plant Mol Biolmosaic virus 18:1027-1029 Potato virus  8 kDa potato Rupasov et al. 1994J Gen Virol X 70:1861-1869 Barley stripe 17 kDa barley Gustafson et al.1986 Nucl Acids Res mosaic virus 14:3895-3909 Potato mop 21 kDa potatoScott et al. 1994 J Gen Virol top virus 75:3561-3568 Peanut 17 kDapeanut Herzog et al. 1994 J Gen Virol clump virus 75:3147-3155 Beetsoil- 22 kDa Sugar beet Koenig et al. 1996 Virology borne virus216:202-207

The above-described point mutations were realized by conventionalmethods known by the person skilled in the art.

The above mutants containing the point mutation were tested for theirability to promote cell-to-cell movement of a viral mutant (with adysfunctional TGB-3 sequence, preferably a BNYVV mutant with adysfunctional P15 gene) when expressed in trans from a replicon. Thesemutants were incapable of promoting such movement and were tested fortheir ability to inhibit infection with a co-inoculated wild type TGB-3virus, preferably co-inoculated with a wild type BNYVV, when the mutantform of the TGB-3 sequence, preferably the P15 gene, was expressed froma replicon.

The inventors have discovered unexpectedly that the genetic modificationmethod according to the invention (preferably a point mutation) could beused to obtain a modified TGB-3 viral sequence (preferably a modifiedBNYVV P15 sequence), which is able to block virus infection withoutproducing deleterious effects when incorporated in the genome of a plantor a plant cell.

It is meant by “being able to block viral infection into a plant or aplant cell,” the possibility to obtain a high degree of tolerance by theplant or plant cell transformed by said modified TGB-3 viral sequence tosaid viral infection, in particular the possibility to ensure rapid andtotal blocking of the virus multiplication and diffusion mechanisms intothe plant, preferably the blocking of the BNYVV virus multiplication anddiffusion mechanisms into a sugar beet plant (beta vulgaris), includingfodder beet, Swiss Chard and table beet which may also be subjected tosaid BNYVV infection.

Said tolerance or resistance could be easily measured by various methodswell known by the person skilled in the art.

Preferably, the genetic modifications in the TGB-3 wild type viralsequence are point mutations in the portions of said wild type viralsequence involved in the mechanisms of viral cell-to-cell movements.

The present invention is also related to the modified TGB-3 viralnucleotide and amino-acid sequences obtained (recovered) by said(modification and selection) method, more preferably the BNYVV P15modified nucleotide and amino-acid sequences obtained (recovered) bysaid method.

Preferably, said BNYVV P15 nucleotide and amino-acid sequences areselected from the group consisting of the following nucleotide (SEQ IDNOs: 1, 3 and 5) or corresponding amino-acid sequences (SEQ ID NOs: 2, 4and 6): SEQ ID NO 1ATGGTGCTTGTGGTTGCAGTAGCTTTATCTAATATTGTATTGTACATAGTTGCCGGTTGT 60 SEQ IDNO 2: M  V  L  V  V  A  V  A  L  S  N  I  V  L  Y  I  V  A  G  CGTTGTTGTCAGTATGTTGTACTCACCGTTTTTCAGCAACGATGTTAAAGCGTCCAGCTAT 120V  V  V  S  M  L  Y  S  P  F  F  S  N  D  V  K  A  S  S  YGCGGGAGCAATTTTTAAGGGGAGCGGCTGTATCATGGACAGGAATTCGTTTGCTCAATTT 180A  G  A  I  F  K  G  S  G  C  I  M  D  R  N  S  F  A  Q  FGGGAGTTGCGATATTCCAAAGCATGTAGCCGAGTCCATCACTAAGGTTGCCACCAAAGAG 240G  S  C  D  I  P  K  H  V  A  E  S  I  T  K  V  A  T  K  ECACGATGTTGACATAATGGTAAAAAGGGGTGAAGTGACCGTTCGTGTTGTGACTCTCACC 300H  D  V  D  I  M  V  K  R  G  E  V  T  V  R  V  V  T  L  TGAAACTATTTTTATAATATTATCTAGATTGTTTGGTTTGGCGGTGTTTTTGTTCATGATA 360E  T  I  F  I  I  L  S  R  L  P  G  L  A  V  F  L  F  M  ITGTTTAATGTCTATAGTTTGGTTTTGGTATCATAGATAA 399C  L  M  S  I  V  W  F  W  Y  H  R  * SEQ ID NO 3:ATGGTGCTTGTGGTTAAAGTAGATTTATCTAATATTGTATTGTACATAGTTGCCGGTTGT 60 SEQ IDNO 4: M  V  L  V  V  K  V  D  L  S  N  I  V  L  Y  I  V  A  G  CGTTGTTGTCAGTATGTTGTACTCACCGTTTTTCAGCAACGATGTTAAAGCGTCCAGCTAT 120V  V  V  S  M  L  Y  S  P  F  F  S  N  D  V  K  A  S  S  YGCGGGAGCAATTTTTAAGGGGAGCGGCTGTATCATGGCCGCGAATTCGTTTGCTCAATTT 180A  G  A  I  F  K  G  S  G  C  I  M  A  A  N  S  F  A  Q  FGGGAGTTGCGATATTCCAAAGCATGTAGCCGAGTCCATCACTAAGGTTGCCACCAAAGAG 240G  S  C  D  I  P  K  H  V  A  E  S  I  T  K  V  A  T  K  ECACGATGTTGACATAATGGTAAAAAGGGGTGAAGTGACCGTTCGTGTTGTGACTCTCACC 300H  D  V  D  I  M  V  K  R  G  E  V  T  V  R  V  V  T  L  TGAAACTATTTTTATAATATTATCTAGATTGTTTGGTTTGGCGGTGTTTTTGTTCATGATA 360E  T  I  F  I  I  L  S  R  L  F  G  L  A  V  F  L  F  M  ITGTTTAATGTCTATAGTTTGGTTTTGGTATCATAGATAA 399C  L  M  S  I  V  W  F  W  Y  H  R  * SEQ ID NO 5:ATGGTGCTTGTGGTTAAAGTAGATTTATCTAATATTGTATTGTACATAGTTGCCGGTTGT 60 SEQ IDNO 6: M  V  L  V  V  K  V  D  L  S  N  I  V  L  Y  I  V  A  G  CGTTGTTGTCAGTATGTTGTACTCACCGTTTTTCAGCAACGATGTTAAAGCGTCCAGCTAT 120V  V  V  S  M  L  Y  S  P  F  F  S  N  D  V  K  A  S  S  YGCGGGAGCAATTTTTAAGGGGAGCGGCTGTATCATGGACAGGAATTCGTTTGCTCAATTT 180A  G  A  I  F  K  G  S  G  C  I  M  D  R  N  S  F  A  Q  FGGGAGTTGCGATATTCCAAAGCATGTAGCCGAGTCCATCACTAAGGTTGCCACCAAAGAG 240G  S  C  D  I  P  K  H  V  A  E  S  I  T  K  V  A  T  K  ECACGATGTTGACATAATGGTAAAAAGGGGTGAAGTGACCGTTCGTGTTGTGACTCTCACC 300H  D  V  D  I  M  V  K  R  G  E  V  T  V  R  V  V  T  L  TGAAACTATTTTTATAATATTATCTAGATTGTTTGGTTTGGATGATTTTTTGTTCATGATA 360E  T  I  F  I  I  L  S  R  L  F  G  L  D  D  F  L  F  M  ITGTTTAATGTCTATAGTTTGGTTTTGGTATCATAGATAA 399C  L  M  S  I  V  W  F  W  Y  H  R  *

In the following description, the various modified BNYVV TGB-3 sequenceswill be hereafter called “P15 mutants,” identified by the followingreference: BNP15-Ala1, corresponding SEQ ID NO: 1 and SEQ ID NO: 2,BNP15-Ala4 corresponding to SEQ ID NO: 3 and SEQ ID NO: 4, BNP15-Asp9,corresponding to SEQ ID NO: 5 and SEQ ID NO: 6.

The nucleotide and corresponding amino-acid sequences of SEQ ID NO: 1,SEQ ID NO: 3 and SEQ ID NO: 5 can be compared to SEQ ID NO: 7 and 8,which are the sequences of the wild type P15 nucleotide and amino-acidsequence already described (Bouzoubaa et al. 1986 J Gen Virol67:1689-1700).

The present invention is also related to the vector comprising saidmodified nucleotide sequence possibly being operably linked to one ormore regulatory sequence(s) active in a plant or a plant cell.Preferably, said vector is a plasmid comprising already said regulatorysequence(s) active in a plant or a plant cell.

The present invention is also related to a method for inducing aresistance to a virus comprising TGB-3 sequence, preferably one of theviruses described in the enclosed Table 1, and more preferably the BNYVVvirus, said method comprising the following steps:

-   -   preparing a nucleic acid construct comprising a nucleic acid        sequence being genetically modified according to the method of        the invention and being operably linked to one or more        regulatory sequences active into a plant or a plant cell,    -   transforming the plant cell with the nucleic acid construct, and    -   possibly regenerating the transgenic plant from the transformed        plant cell.

Preferably, said method is used for inducing a resistance to the BNYVVinto a sugar beet plant or a sugar beet cell. Said method comprises thefollowing steps:

-   -   preparing a nucleic acid construct comprising a modified nucleic        acid sequence obtained by the method according to the invention,        preferably preparing a nucleic acid construct comprising a        nucleic acid sequence selected from the group consisting of SEQ        ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, being operably linked to        one or more regulatory sequences active into a plant,    -   transforming the sugar beet plant cell with the nucleic acid        construct, and    -   possibly regenerating the transgenic sugar beet plant from the        transformed sugar beet plant cell.

The present invention is also related to the obtained (recovered)transgenic plant or the transgenic plant cell resistant to an infectionby a virus comprising a TGB-3 sequence, preferably one of the virusesdescribed in the enclosed Table 1, more preferably the BNYVV virus, saidplant or plant cell comprising a nucleic acid construct having a TGB-3modified nucleic acid sequence, being operably linked to one or moreregulatory sequences capable of being active in a plant or a plant cell.

Preferably, said modified nucleic acid sequence is selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, beingoperably linked to one or more regulatory sequences active in a plant ora plant cell.

Preferably, the cell is a stomatal cell and the regulatory sequencecomprises a promote sequence and a terminator sequence capable of beingactive in a plant. Said promoter sequence can be constitutive or couldbe obtained from a foreign promoter sequence, and is preferably selectedfrom the group consisting of the 35S Cauliflower Mosaic Virus promoter,and/or the polyubiquitin Arabidopsis thaliana promoter.

Advantageously, the promoter sequence is a promoter which is mainlycapable of being active in the root tissue of plants such as the parpromoter or the hemoglobin gene from Perosponia andersonii.

A last aspect of the present invention is related to a transgenic planttissue such as fruit, stem, root, tuber, seed of the transgenic plantaccording to the invention or a reproducible structure (preferablyselected from the group consisting of calluses, buds or embryos)obtained from the transgenic plant or the plant cell according to theinvention.

The techniques of plant transformation, tissue culture and regenerationused in the method according to the invention are the ones well known bythe person skilled in the art. Such techniques are preferably the onesdescribed in the International Patent Applications WO 95/101778, WO91/13159 (corresponding to the European Patent ApplicationEP-B-0517833), WO 98/07875, which are incorporated herein by reference.

These techniques are preferably used for the preparation of transgenicsugar beet plants and plant cells according to the invention.

1. A method of identifying mutants in a triple gene block 3 (TGB-3)viral sequence which inhibit infection of a virus into a cell,comprising: mutating said TGB-3 sequence; selecting TGB-3 mutants whichno longer promote cell-to-cell movement of a (TGB-3 minus) mutant viruswhen expressed in trans from a replicon; further selecting from theidentified mutants those which also inhibit infection with aco-inoculated wild type virus when the mutant TGB-3 is expressed from areplicon; recovering said mutant TGB-3 viral sequence wherein a mutantTGB-3 sequence recovered from the foregoing selection steps is a mutantTGB-3 viral sequence that inhibits infection of a virus into a cell. 2.The method according to claim 1, wherein the TGB-3 wild type viralsequence is the beet necrotic yellow vein virus (BNYVV) P15 sequence. 3.A genetically modified TGB-3 viral sequence obtained by the method ofclaim
 1. 4. The genetically modified TGB-3 viral sequence according toclaim 3, selected from the group consisting of: SEQ ID NOS: 1, 3, and 5.5. A vector comprising the genetically modified TGB-3 viral sequenceaccording to claim
 3. 6. A method for inducing resistance to a virus ina plant or a plant cell comprising: preparing a nucleic acid constructcomprising a genetically modified TGB-3 viral sequence according toclaim 3 operably linked to one or more regulatory sequence(s) active ina plant or a plant cell, and transforming a plant cell with said nucleicacid construct.
 7. The method according to claim 6, wherein the virus isselected from the group consisting of the apple stem pitting virus, theblueberry scorch virus, the potato virus M, the white clover mosaicvirus, the Cymbidium mosaic virus, the barley stripe mosaic virus, thepotato mop top virus, the peanut clump virus, the beet soil-borne virusand the BNYVV virus.
 8. The method according to claim 6 wherein theplant cell is a stomatal cell.
 9. The method according to claim 6wherein the plant is selected from the group consisting of apple,blueberry, potato, clover, orchid, barley, peanut and sugar beet. 10.The method according to claim 6, wherein the regulatory sequencecomprises a promoter sequence or a terminator sequence active in aplant.
 11. The method according to claim 10, wherein the promotersequence is a constitutive or a foreign promoter sequence.
 12. Atransgenic plant or transgenic plant cell resistant to a viruscomprising a nucleic acid construct having a genetically modified TGB-3viral sequence according to claim 4 operably linked to one or moreregulatory sequence(s) active in a plant or a plant cell.
 13. Atransgenic plant or transgenic plant cell according to claim 12, whereinthe virus is selected from the group consisting of the apple stempitting virus, the blueberry scorch virus, the potato virus M, the whiteclover mosaic virus, the Cymbidium mosaic virus, the potato virus X, thebarley stripe mosaic virus, the potato mop top virus, the peanut clumpvirus, the beet soil-borne virus and the BNYVV virus.
 14. The transgenicplant or transgenic plant cell according to claim 12 selected from thegroup consisting of apple, blueberry, potato, clover, orchid, barley,peanut and sugar beet.
 15. The transgenic plant or transgenic plant cellaccording to claim 12, wherein the regulatory sequence comprises apromoter sequence and a terminator sequence active in a plant.
 16. Thetransgenic plant or transgenic plant cell according to claim 12, whereinthe regulatory sequence(s) comprise a promoter sequence which is aconstitutive or a foreign vegetal promoter sequence.
 17. The transgenicplant or transgenic plant cell according to claim 16, wherein thepromoter sequence is selected from the group consisting of the 35SCauliflower Mosaic Virus promoter, the polyubiquitin Arabidopsisthaliana promoter, and both.
 18. The transgenic plant or transgenicplant cell according to claim 16 wherein promoter sequence is active inroot tissues.
 19. The transgenic plant tissue of claim 12 wherein saidtissue is selected from the group consisting of fruit, stem, root,tuber, and seed.
 20. The vector of claim 5 operably linked to one ormore regulatory sequence(s) active in a plant cell.